Neuronal cell death resulting from ischemic brain injury may involve apoptotic mechanisms, especially when the ischemic insult is relatively mild and cellular energy metabolism is not severely compromised. Current studies have identified a group of effector caspases, particularly caspase-3 (and probably caspase-7), as the central executive molecules in apoptotic ischemic neuronal death. Caspase-3 is initially synthesized in cells as inactive zymogen. Sequence-specific proteolytic cleavage converts the caspase-3 zymogen to its active form, whichsubsequently cleaves various substrates, leading to apoptosis. Activation of caspase-3 may involve both intrinsic (Apaf-1/caspase-9 apoptosome dependent) and extrinsic (cell membrane receptor mediated) pathways. However, the precise mechanism by which caspase-3 is activated in ischemic neurons is poorly understood. Thus, the objective 1 of this proposal is to define the upstream signaling pathway leading to caspase-3 activation after cerebral ischemia. The hypothesis underlying this line of research is that formation of the Apaf-1/caspase-9 apoptosome plays a central role in mediating caspase-3/7 activation and ischemic neuronal cell death. Under certain experimental conditions, blockage of caspase activities is able to delay, but not prevent completely, neuronal apoptosis, suggesting that caspase-independent death pathways must be involved. A novel pro-apoptotic molecule, designated as AIF (apoptosis-inducing factor), has now been identified. AIF, which isactivated and released from the mitochondria upon receiving cell death signals, potently promotes nuclear apoptosis, independent of any caspase activities. Our preliminary studies strongly suggest that AIF is activated in neurons after cerebral ischemia and mediates neuronal apoptosis, in addition to caspase activation. Thus, the objective 2 of this proposal is to define the role of AIF in ischemic neuronal cell death. The hypothesis underlying this research is that AIF-dependent and caspase-dependent mechanisms contribute independently and synergistically to neuronal cell death after cerebral ischemia. We propose the following specific aims to test the hypotheses: Aim 1. Determine the role of the formation of Apaf-1/caspase-9 apoptosome in caspase-3 activation andhippocampal CA1 neurodegeneration after transient cerebral ischemia. Aim 2. Determine the role of synergistic action of AIF-dependent and caspase-dependent pathways inmediating hippocampal CA1 neurodegeneration after transient cerebral ischemia. Aim 3. Determine the role and mechanisms by which AIF is activated after ischemia using neuronalculture models. These studies will take advantage of our recent cloning of the rat genes encoding novel dominant-negative inhibitory proteins for caspase-9, Apaf-1 and AIF. The deduced AIF and apoptosome pathways will be confirmed in both in vivo and in vitro models of cerebral ischemia by the powerful and noncytotoxic AAV (adeno-associate virus vector)-mediated gene infection of the dominant-negative proteins.